The invention relates to a class of peptide and peptide-like compounds, xe2x80x9cBuforninsxe2x80x9d which inhibit the enzymatic activity of Botulinum toxin B and Tetanus neurotoxins.
The Botulinum toxins (Bttxs) are among the most potent toxins to animals, e. g. the LD50 in mice is about 1 ng/kg. Bttxs comprise a family of seven distinct serotypes (A-G). Bttxs are composed of two subunits comprising a 100 kdal nerve-cell target heavy chain and a 50 kdal endoproteolytically active light chain. These toxins are Zn-metalloproteases and contain a Zn-protein binding motif HEXXH.
However, Zn-metalloprotease inhibitors, such as angiotensin converting enzyme inhibitors, captopril and phosphoramidon, are not effective inhibitors of Bttxs. Although Zn-chelators inhibit Bttx protease activity in vitro, they merely delay the protease activity in vivo and in tissue preparations comprising intact nerve and muscles cells and/or tissues. Furthermore, some Zn-chelators are toxic at concentrations necessary to delay the Bttx protease activity. Although dithiocarbamates inhibit other Zn-containing proteins such as SOD, they are ineffective against the Bttx serotype B (BttxB). Clearly, inhibitors of the various Bttx serotypes, such as BttxB, are needed.
BttxB specifically cleaves synaptobrevin (VAMP2) between glutamine 76 and phenylalanine 77 (QF bond or cleavage site). There is an obligatory requirement for a relatively long substrate for the in vivo target VAMP2 as shown by efforts to produce a minimum length substrate. It has been shown that 30 amino acids of VAMP2 are required and 40 amino acids of VAMP2 are required for optimum cleavage. See Shone, C. C. et al. (1993) Eur. J. Biochem 217:965-971. V2, a peptide derived from VAMP2, is a sequence of 10 amino acids located 4 residues upstream from the cleavage site, and was found to inhibit Bttx activity. See Pellizzari R. et at. (1996) J. Biol. Chem. 271:20353-20358. In VAMP2, a mutation of the C-terminal amino acids had little effect; whereas a helix disrupting substitution of Pro for Ala inhibited BttxB activity by 28%. Further, replacement of several negatively charged amino acids led to almost complete inacivity. See Whitcome, M, et al. (1996) FEBS Let. 386:133-136).
Computer-aided secondary structure analysis of VAMP2 predicted two stretches of xcex1-helical structure flanking the cleavage site QF. See Witcome, M. R. et al. (1996) FEBS Let. 386: 133-136. Computer-aided tertiary structure analysis indicates that the two helices could self associate to form a supersecondary structure of a helix bundle with the helices separated by a reverse turn. See Lebeda F. J., et al. (1996) Med. Defense Biosci. Rev. 204.
The above results indicate that more than just the QF bond is required to be recognized by the toxin for substrate cleavage.
Recently, a new class of compounds have been discovered, which have a characteristic conformation and the QF bond, that inhibit the Bttx protease activity. These compounds and their uses are disclosed herein below.
The invention is directed to a class of peptides and peptide-like compounds, Buforinins, which have an internal QF bond and the ability to inhibit BttxB protease activities. As the tetanus toxin cleavage site is the same as BttxB, Buforinins may also competitively inhibit tetanus protease activity.
Thus, in one aspect, the invention is directed to compounds of the formula:
X1X2B3X4B5X*6X7X8
B9X10B11X12B13X14
B15X16B17X*18X*19B20X21
X22X23Q24F25Z*26X27
X28B29X30B31B32X33X34
B35B36X37Z38Z39xe2x80x83xe2x80x83(1)
and the salts, esters, amides, and acyl forms thereof. Up to 15 amino acids may be truncated from the N-terminus and up to 6 amino acids may be truncated from the C-terminus. Each position represented by a letter indicates a single amino acid residue wherein B is a basic or polar/large amino acid or a modified form thereof; X is a small or hydrophobic amino acid or a modified form thereof; X* is a small or polar/large amino acid or a modified form thereof; Z is a polar/large or hydrophobic amino acid or a modified form thereof; Z* is Proline or a polar/large or hydrophobic ammo acid or a modified form thereof. As described below, one or more of the peptide linkages between the amino acid residues may be replaced by a peptide linkage mimic.
In other a the invention is directed to recombinant materials useful for the production of those peptides of the invention that contain gene-encoded amino acids, as well as plants or animals modified to contain expression systems for the production of these peptides. The invention also includes methods to prepare and manipulate these recombinant materials.
In addition, the invention is directed to pharmaceutical compositions containing the compounds of the invention as active ingredients and to compositions which contain expression systems for the production of the peptides. The invention is also directed to methods to prepare the invention compounds synthetically, to antibodies specific for these compounds, and to the use of the compounds as preservatives, therapeutics, and prophylactics.
The invention is also directed to the use of the compounds of the invention in assays for detection of BttxB and Tttx by the use of selective inhibition and for determining inhibitors and substrates for a given toxin.
The present invention relates to materials, compositions, kits and methods for inhibiting the enzymatic activity of Botulinum toxin B and Tetanus neurotoxins.
The invention further relates to materials, compositions, kits and methods for preventing or treating toxic poisoning such as Botulinum toxin B and tetanus poisoning. The kits can an provide single or multiple dosage and can include other conventional ancillary materials such as instructions, solutions and compositions needed for operation. The compositions and solutions may be placed in containers, lest tubes, etc. Containers could be similar to those employed in insect/snake bite kits that includes an injector which provides the Buforinin, and TCEP in separate chambers. If chaotropes are present, they are separately included in one or more containers.
A kit for determining whether a sample contains a Buforinin, the amount of said Buforinin or the type of said Buforinin may include antibodies immunospecific for Buforinins.
A kit for determining whether a sample contains a Botulinum toxin or the type of the Botulinum toxin may include antibodies immunospecific for at least one Buforinin having an interaction with a Botulinum toxin. Likewise, a kit for determining whether a sample contains a Tetanus toxin would include antibodies immunospecific for at least one Buforinin having an interaction with a Tetanus toxin.
Another embodiment includes Buforin I along with one or more known peptide inhibitors associated with the decontamination of Botulinum B and/or Tetanus toxins. Additionally, the kits may also include a stable peptide mixture or powder which includes Buforinin for sprinkling over food or wounds for detoxification.